Sex types of papaya are controlled by a pair of nascent sex chromosomes, but molecular genetic mechanisms of sex determination and sex differentiation in papaya are still unclear. We performed comparative analysis of transcriptomic profiles of male and female floral buds at the early development stage before the initiation of reproductive organ primordia at which there is no morphological difference between male and female flowers. A total of 1734 differentially expressed genes (DEGs) were identified, of which 923 showed female-biased expression and 811 showed male-biased expression. Functional annotation revealed that genes related to plant hormone biosynthesis and signaling pathways, especially in abscisic acid and auxin pathways, were overrepresented in the DEGs. Transcription factor binding motifs, such as MYB2, GAMYB, and AP2/EREBP, were enriched in the promoters of the hormone-related DEGs, and transcription factors with those motifs also exhibited differential expression between sex types. Among these DEGs, we also identified 11 genes in the non-recombining region of the papaya sex chromosomes and 9 genes involved in stamen and carpel development. Our results suggested that sex differentiation in papaya may be regulated by multiple layers of regulation and coordination and involved transcriptional, epigenetic, and phytohormone regulation. Hormones, especially ABA and auxin, transcription factors, and genes in the non-recombination region of the sex chromosome could be involved in this process. Our findings may facilitate the elucidation of signal transduction and gene interaction in sex differentiation of unisexual flowers in papaya.

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